Rheumatoid arthritis is a chronic inflammatory disease involving multiple joints. The main pathology of the affected synovial tissue consists of the hyperplasia and the subintimal infiltration of T and B lymphocytes. Such inflammation of the synovial tissue is thought to be caused by T lymphocyte reactive to an unknown autoantigen. Nonetheless, the T lymphocyte infiltrated in the almost tissues does not show any indication of activation on the surface of cell and also does not almost express cytokines. In contrast to this, it is observed that both synovial tissue and fluid are enriched with the cytokines derived from macrophage. These cytokines may include interleukin-1 (IL-1) which can accelerate the growth of synovial fibroblast and tumor necrosis factors (TNFs). These experimental results suggest the hypothesis that T lymphocyte is importantly associated with the induction of inflammation to synovial tissues and the inflammation is maintained by the cytokines derived from the activated synovial cells.
One of the major intents of rheumatoid arthritis treatment is to prevent the synovial tissue hyperplasia, because it forms the pannus tissue that irreversibly destroys the cartilage and bone in the affected joint. Effective drugs for treating rheumatoid arthritis have not been developed until the present time and the developed drugs can exhibit limited efficacies. Once arthritis occurs, it causes economic loss as well as severe pain.
Medical treatments of rheumatoid arthritis being used presently are as follows. The drugs used often for initial treatment are non-steroidal anti-inflammatory drugs (NSAIDs). These NSAIDs limitedly improve a patient's condition, but cannot prevent the cartilage destruction of joint area or the progress of disease. Moreover, half the patients using this treatment should stop the treatment within one year because of serious side effect. Next, gold drugs such as gold sodium thiomalate and gold sodium thiosulfate, or disease modifying anti-rheumatic drugs (DMARDs) such as penicillamine and anti-malarials are used. These drugs also decrease the progress of rheumatoid arthritis, but after 5 years of the treatment using DMARDs, only 5–15% of the patients adhere to use the drugs because the serious side effect can be accompanied. If the drugs mentioned above are not effective any more, the affected joint area with rheumatoid arthritis should be replaced by artificial joint by surgical operation.
In this manner, most of the treatments of rheumatoid arthritis used until now were not designed with a particular target molecule and had a limitation of showing slight effects in most cases. In the meantime, it has been reported that therapeutic effects appeared by taking notice of inflammation-inducing cytokines such as TNF as a target molecule for treating the rheumatoid arthritis and introducing an antibody specific to TNF or a soluble TNF-receptor into the affected area of a patient to result in inactivation of the TNF (Maini, R. N. et al., Immunol. Rev. 144:195, 1995; Moreland, L. W. et al., N. Engl. J. Med. 337:141, 1997). In company with this, various gene transfer experiments in vivo are progressing in an animal model of rheumatoid arthritis with continuously expressing the molecules having an immune inhibitory function. Consequently, most of the current treatments are directed to correction of the immune aberration that supposedly drives the synovial cell proliferation.
Angiogenesis, the formation of new blood vessels, is one of the earliest histopathologic findings in rheumatoid arthritis and appears to be required for pannus development. This neovascularization is thought not only to maintain the chronic architectural changes via delivery of required blood-borne elements to the pannus, but also to play an active role in inflammation as a source of both cytokine and protease activity. The expanded vascular-bed volume resulting from angiogenesis may provide increased access for inflammatory cells to infiltrate the synovium. Although the factors specifically promoting angiogenesis in rheumatoid arthritis have not yet been identified, both synovial tissue and fluid are enriched with angiogenesis-promoting molecules. These include cytokines, such as basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and soluble adhesion molecules, such as E-selectin. These data suggested a therapeutic potential for using an anti-angiogenic procedure for favorably changing the disease course of rheumatoid arthritis.
Until now a lot of factors that repress angiogenesis have been found. Most of them are created from the cleavage of protoprotein, and representatively angiostatin, endostatin and platelet factor-4 and the like have been known.
Angiostatin is composed of 98th to 440th amino acids of plasminogen. Angiostatin was initially isolated from mice bearing a Lewis lung carcinoma and was identified as a 38-kDa internal fragment of plasminogen that encompasses the first four kringles of the molecule (O'Relly, M. et al., Cell 79:715, 1994). It was reported that the growth of primary tumors was inhibited effectively by injecting purified angiostatin hypodermically in six cancer model experiments (O'Relly, M. et al, Nat. Med 2:689, 1996).
Endostatin consists of C-terminal 183 amino acids of collagen XVIII and has an anti-angiogenic activity. It was reported that the growth of primary tumors was inhibited effectively by injecting purified endostatin hypodermically in four cancer model experiments (O'Relly, M. et al., Cell 88:277, 1997).
Platelet factor-4 belongs to CXC cytokine family, which consists of chemotactic polypeptides below 10 kDa, and has an anti-angiogenic activity. It was reported that the platelet factor-4 inhibited the growth of cancer, such as B-16 melanoma and HCT-116 colon carcinoma (Maion, T. E. et al., Cancer Research, 51:2077, 1991).
Angiogenesis is known to be associated with various diseases, such as tumor formation and metastasis, retinitis, angioma, chronic inflammation, intestinal adhesions, atherosclerosis, rheumatoid arthritis and so on, but it has not yet been verified that anti-angiogenic factors were effective to all the diseases listed above actually. Only the tumor inhibitory effects of these factors associated with a particular disease have been reported (U.S. Pat. No. 5,856,315, U.S. Pat. No. 5,733,876, U.S. Pat. No. 5,792,845, U.S. Pat. No. 5,854,205, U.S. Pat. No. 6,024,688). The US patents disclose that the treatment effects for various kinds of diseases, such as ovarian carcinoma (HTB161, A2780S), colon carcinoma (MIP, CACO2), Lewis Lung Carcinoma (LLC), fibrosarcoma (T241), prostate gland carcinoma (PC-3) and breast carcinoma (MDA-MB), were identified by injecting anti-angiogenic factors with the type of recombinant protein.
Meanwhile, unlike general treating method that applies a toxicity to cells directly, the treating method, which cures diseases by inhibiting angiogenesis by means of supplying anti-angiogenic factors as described above, is based on the principle of inhibiting the cell growth, so anti-angiogenic factors over certain concentration should be supplied continuously to exhibit effects in vivo. But, the method of supplying anti-angiogenic factors with the type of recombinant proteins costs too much for administering proteins continuously, is troublesome and has a problem in that it imparts toxicity to a patient. Therefore, it has been required to develop a method of supplying anti-angiogenic factors to the affected area continuously and locally.
Accordingly, the present inventors have attempted new approaches for the probability of the treatment of rheumatoid arthritis by anti-angiogenesis in order to replace the prior treatment of rheumatoid arthritis having focused on solving the immunological problems. More particularly, the present inventors obtained the cell lines for producing representative anti-angiogenic factors such as angiostatin, endostatin and platelet factor-4 through inserting their genes into a viral vector and then transplanting them the affected area of mice induced with rheumatoid arthritis. We also performed the histological examinations for the level of hyperplasia in synovial cell and cartilage destruction and the immunological examinations for the concentrations of cytokines associated with the joint inflammation, as well as macroscopic examination for joint swelling to obtain the results for the progressive level of rheumatoid arthritis. The results showed that the incidence of rheumatoid arthritis in our treatment was remarkably reduced in comparison with the control group. This is to show that our gene therapy using an anti-angiogenic gene is effective to treatment of rheumatoid arthritis.